Fig 4. Suboptimal spacing of the P_sigG -10 and -35 elements diminishes sigG expression.

(A) Alignment of ten B. subtilis promoters activated by σ^F, including P_sigG. Nucleotides comprising the -10 and -35 elements of each promoter are in bold and shaded gray. Transcription start sites, if known, are underlined [41]. The consensus promoter sequence for σ^F is shown above [38]. Red arrowheads indicate two notable features of P_sigG that differ from the other σ^F-target promoters: shorter spacing of the -10 and -35 promoter elements (14 nt as opposed to the more common 15 nt; left arrowhead), and a T at position -7 (more typically an A or G; right arrowhead). (B, C) P_sigG activation is significantly stimulated by increasing the spacing between the -10 and -35 elements to 15 nt. β-Galactosidase production was monitored during sporulation of strains harboring P_sigG-lacZ (WT [14 nt]; open circles) and ^15ntP_sigG-lacZ (15 nt; closed circles), a variant in which a single nucleotide was inserted between the P_sigG -10 and -35 elements to increase their spacing to 15 nt. (Strains JJB31 and JJB51, respectively.) Note that (C) provides a zoomed view of the data from the boxed area of (B). (D, E) The T at position -7 at most only modestly influences P_sigG activation. The activity of P_sigG-lacZ (WT; closed circles),^T→AP_sigG-lacZ (T→A; closed diamonds), and ^T→GP_sigG-lacZ (T→G; open diamonds) was measured during sporulation. (Strains JJB31, JJB87, and JJB89, respectively.) The T→A and T→G variants were engineered to harbor an A or G, respectively, at position -7 in place of T. Note that (E) provides a zoomed view of the data from the boxed area of (D). The timing of the σ^F-to-σ^G switch, between sporulation hours 2.5 and 3, is indicated in each panel by a dashed gray line. For all relevant panels, error bars indicate ± standard deviations based on three independent experiments. *p < 0.05, **p < 0.01 Student’s t-test.